(1) Field of the Invention
This invention relates to a mature bone morphogenetic protein of which some hydrophobic amino acid residues are exchanged to a hydrophilic or a polar amino acid residue by chemical modification or genetic engineering technology. The mature proteins of this invention show an antagonistic activity against bone morphogenetic proteins, and are useful for medicinal agents to suppress symptoms of ectopic osteogenesis and ectopic calcification or metabolic bone diseases with calcification such as neurotic osteosis, ectopic ossification caused by stress of operation, traumatic myositis ossificans, ossification by defect of oxygen supply, osteogenic tumor, ossification of the posterior longitudinal ligament, and arterial sclerosis.
(2) Description of the Related Art
A bone morphogenetic protein (hereinafter called BMP) is a protein having a bone morphogenetic activity in decalcified bone tissue. Although the isolation of BMP had been worked on energetically since the 1970s, it was quite difficult to isolate as a single protein. Gene cloning of BMP as expected was performed by Wozney in 1989 by molecular biological technology, using the amino acid sequences derived from unknown peptides which were separated by treating the fraction having bone morphogenetic activities with an enzyme. The gene was immediately introduced to the animal cultured cells, and the activity of the protein expressed was measured in vivo, and BMP activity in the protein was practically proved (Wang, E. A. et al., (1990) Proc. Natl. Acad. Sci. USA, vol. 87, p. 2220–2224). Continuing cloning a protein with bone morphogenetic activities utilizing homology, several numbers of the proteins with bone morphogenetic activities in a similar structure have been isolated so far. Those proteins all belong to TGF (transforming growth factor)-β superfamily and are proved to have the activity to cause ectopic ossification in vivo, basically. Ossification caused by BMP is said to be internal cartilaginous and it seems to reproduce the formation of long bone at an embryonal stage. Therefore, BMP itself can be used as a medicinal agent for the treatment to compensate the bone deficit.
On the other hand, since BMP genes were disclosed and the specific antibodies against BMPs were prepared, BMPs were also expressed at the site of ectopic calcification, which have not had any medical treatment so far, and there seem some possibilities of the relationship between BMPs and those diseases. For example, it becomes recently evident that BMP exists or is included in diseases such as neurotic osteosis, ectopic ossification caused by stress of operation, traumatic myositis ossificans, ossification by defect of oxygen supply, osteogenic tumor, specified as refractory diseases such as ossification of the posterior longitudinal ligament (OPLL) (Spine, 17-3S, S33, 1992) and calcification part of arterial sclerosis (J. Clin. Invest., vol. 91, p. 1800, 1993). In addition, the major symptoms of pseudomalignant heterotopic ossification (PHO), pseudomalignant osseous tumor and myosistis ossificans circumscripta are ache and the appearance of the hard tissue mass in the muscle. Though the causes of these diseases are still unknown in detail, BMP seems to have a relationship with the appearance of hard tissue in the muscle of the patients. It is considered that BMP exists in the tissue in which BMP does not exist naturally, acts on autocrine, and forms bone like hard tissue. There is no effective treatment for OPLL by now. When the compressive neural symptom is critical, an excision is operated. However the prognosis is not so good. There is no treatment for calcification of artery, neither.
It seems that suppression of BMP existence may be one of the major treatments for these diseases. Another treatment, such as administration of BMP antagonists, also seems effective. BMP receptors, neutralized antibodies against BMP and the BMP peptides corresponding to the binding site, a BMP with chemically modified specific amino acid residue are thought to have BMP antagonist-like activity.
Many studies have been so far carried out concerning a relationship between the structure and the activity of BMPs and it is speculated that some sites of mature BMPs relate to the receptor binding. It is known that a peptide synthesized based on these studies works as an antagonist against BMPs (JP patent application, Hei 7 ('95)-200175).